Colorimeter , Spectrophotometer

and Mass Spectrometer.

By: Mr. Prachand Man Singh Rajbhandari.BSc Medical Biochemistry (Nobel College, Pokhara University, Nepal)

MSc Medical Biochemistry (JN Medical College, KLE University, Belgaum)

Outlines

• Introduction

• Principle

• Laws

• Flow representation

• Instrumentation

• Applications

• Mass spectrometer

- Principle

- Instrumentation

INTRODUCTION

COLORIMETER

• Colorimeter is instrument which is used in the

measurement of the luminious intensity of

light.

• Invented by Louis Jules Duboscq in 1870.

PRINCIPLECOLORIMETER

• Involves the quantitative estimation of colors.

• The difference in color intensity results in the

difference in the absorption of light.

• The intensity of color is directly proportional

to the concentration of the compound being

measured.

CONTD.

• The amount of light absorbed or transmitted by

a colored solution is in accordance with two

laws:

• Beer’s law

• Lambert’s law

LAWS

Beer’s law :

• AαC

Lambert’s law :

• AαL

Io I

Derivation of the Formula

• Combining the two laws

AαCxL

OR A=KxCxL

• Let AT=absorbance of the test solution

• CT=concentration of the test solution

• AS=absorbance of the standard solution

• CS=concentration of the standard solution

2/2/2015 12:20 PM

2/2/2015 12:20 PM

AT

AS

KxCTxL

KxCSxL=

AT

AS

CT

CS=

CT =AT

AS

X CS

AS=KxCSxLAT=KxCTxL

2/2/2015 12:20 PM

CT =AT

AS

X CS

Concentration of TEST solution

Absorbance of TEST

Absorbance of STANDARD

Concn of STANDARDX

=

Concentration of TEST /100ml

Absorbance of TEST

Absorbance of STANDARD

Concn of Std X 100X

=Xml

2/2/2015 12:20 PM

Concentration of TEST /100ml

Absorbance of TEST

Absorbance of STANDARD

X

=Xml

Concn of Std X 100

Concentration of TEST /100ml

O.D of ‘T’- O.D of ‘B’

O.D of ‘S’- O.D of ‘B’

X

=Volume of ‘T’

Amount of ‘S’ X 100

Concentration of TEST /100ml

T - B

S - B

X=Volume of ‘T’

Amount of ‘S’ X 100

Flow representation of colorimeter

Parts of the colorimeter

• Light source

• Slit

• Condensing lenses

• Filter

• Detector (photocell)

• Output :

INSTRUMENTATION

Complementary filters for coloured solutions.

The selected filters has the color to the complementary to that of

the color of unknown solution

• Cuvette are rectangular cell , square cell or circular one

• Made up of optical glass for visible wavelength.

• Common one is square,rectangularto avoid refraction artefacts.

• dimension of cuvette is 1cm.

Cuvette(sample holder)

cuvettes

• For estimation of biochemical samples , like plasma, serum,

cerebrospinal fluid (csf ) , urine.

• Ex. Determination of blood glucose, blood urea, serum

creatinine, serum proteins, serum cholesterol, serum inorganic

phosphate, urine creatinine & glucose in CSF, etc.

• They are used by the food industry and by manufacturers of

paints and textiles.

APPLICATION OF COLORIMETRIC

ASSAY

SPECTROPHOTOMETER

Introduction

• compounds absorb light radiation of a specific wavelength.

• the amount of light radiation absorbed by a sample is

measured.

• The light absorption is directly related to the concentration of

the compound in the sample.

• As Concentration increases, light Absorption increases,

linearly, As Concentration increases, light Transmission

decreases, exponentially.

19

Introduction

• Spectrophotometer:

a) Single-beam.

b) Double-beam

[4]

20

Beer-Lambert law

• Light Absorbance: (A) = log (I0 / I)= ƐLC

• Light Transmission (T) = I/I0 = 10-ƐCL

• I0: Light Intensity entering a sample

• I: Light Intensity exiting a sample

• C: The concentration of analyte in sample

• L: The length of the light path in glass sample cuvette

• Ɛ: a constant for a particular solution and wave length

21

[5]

Flow representation of

spectrophotometer

22

Parts of spectrophotometer

• Light source:.

23

INSTRUMENTATION

Parts of spectrophotometer

• Monochromator : Accepts polychromatic input light from

a lamp and outputs monochromatic light.

24

Parts of spectrophotometer

• Dispersion devices: A special plate with hundreds

of parallel grooved lines.

• The grooved lines act to separate the white light into

the visible light spectrum.

25

The more lines

the smaller

the wavelength

resolution.

Parts of spectrophotometer

• Focusing devices: Combinations of lenses, slits,

and mirrors.

• relay and focus light through the instrument.

26

• Cuvettes: designed to hold samples for spectroscopic

experiments. made of Plastic, glass or optical grade

quartz

• should be as clear as possible, without impurities that

might affect a spectroscopic reading.

27

Parts of spectrophotometer

• Detectors: Convert radiant energy (photons) into an

electrical signal.

The photocell and phototube are the simplest

photodetectors, producing current proportional to the

intensity of the light striking Them .

28

Applications

1. Concentration measurement

– Prepare samples

– Make series of standard solutions of known concentrations

29

Applications

− Measure the absorption of the unknown, and from the

standard plot, read the related concentration

30

Applications

2. Chemical kinetics

• Kinetics of reaction can also be studied using

UV spectroscopy. The UV radiation is passed through

the reaction cell and the absorbance changes can be

observed.

31

MASS SPECTROMETER

(Principle and Instrumentation)

PRINCIPLES

Technique involves

• - Creating gas phase ions from the analyte atoms or molecules

• - Separating the ions according to their mass-to-charge ratio (m/z)

• - Measuring the abundance of the ions

PRINCIPLES

Technique can be used for

• - Qualitative and quantitative analysis

• - Providing information about the mass of atoms and molecules

• - Molecular structure determination (organic & inorganic)

• - Identification and characterization of materials

PRINCIPLES

• - Separates gas phase ionized atoms, molecules, and fragments

of molecules

• - Separation is based on the difference in mass-to-charge ratio (m/z)

• m = unified atomic mass units (u)

• 1 dalton (Da) = 1 u = 1.665402 x 10-27 kg

• z = charge on the ion (may be positive or negative)

PRINCIPLES

• - Analyte molecule can undergo electron ionization

• M + e- → M●+ + 2e-

• - M●+ is the ionized analyte molecule called molecular ion

• - Radical cation is formed by the loss of one electron

• - Permits easy determination of molecular weight of analyte

General Structure of Mass

Spectrometer

References

• Text book of biochemistry,

DM Vasudevan and U. Satyanaarayana

• Principle and techniques of biochemistry and

molecular biology, wilson and walker.

• Hand book of Biomedical Instrumentation. R S

Khandpur

• Clinical chemistry. Bishop.

• Wikipaedia.

THANK YOU..